Organo luminescent semiconductor nanocrystal probes for biological applications and process for making and using such probes

ABSTRACT

A semiconductor nanocrystal compound is described capable of linking to an affinity molecule. The compound comprises (1) a semiconductor nanocrystal capable of emitting electromagnetic radiation and/or absorbing energy, and/or scattering or diffracting electromagnetic radiation—when excited by an electromagnetic radiation source or a particle beam; and (2) at least one linking agent, having a first portion linked to the semiconductor nanocrystal and a second portion capable of linking to an affinity molecule. The compound is linked to an affinity molecule to form a semiconductor nanocrystal probe capable of bonding with a detectable substance. subsequent exposure to excitation energy will excite the semiconductor nanocrystal in the probe causing the emission of electromagnetic radiation. Further described are processes for respectively: making the luminescent semiconductor nanocrystal compound; making the semiconductor nanocrystal probe; and using the probe to determine the presence of a detectable substance in a material.

[0001] The invention described herein arose in the course of, or under,Contract No. DE-AC03-SF00098 between the United States Department ofEnergy and the University of California for the operation of the ErnestOrlando Lawrence Berkeley National Laboratory. The Government may haverights to the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to organo luminescent semiconductornanocrystal probes for biological applications wherein the probesincludes a plurality of semiconductor nanocrystals capable ofluminescence and/or absorption and/or scattering or diffraction whenexcited by a radiation or particle beam.

[0004] 2. Description of the Related Art

[0005] Fluorescent labeling of biological systems is a well knownanalytical tool used in modem bio-technology as well as analyticalchemistry. Applications for such fluorescent labeling includetechnologies such as medical (and non-medical) fluorescence microscopy,histology, flow cytometry, fluorescence in-situ hybridization (medicalassays and research), DNA sequencing, immuno-assays, binding assays,separation, etc.

[0006] Conventionally, such fluorescent labeling involves the use of anorganic dye molecule bonded to a moiety which, in turn, selectivelybonds to a particular biological system, the presence of which is thenidentified by excitation of the dye molecule to cause it to fluoresce.There are a number of problems with such an analytical system. In thefirst place, the emission of light of visible wavelengths from anexcited dye molecule usually is characterized by the presence of a broademission spectrum as well as a broad tail of emissions on the red sideof the spectrum, i.e., the entire emission spectrum is rather broad. Asa result, there is a severe limitation on the number of different colororganic dye molecules which may be utilized simultaneously orsequentially in an analysis since it is difficult to eithersimultaneously or even non-simultaneously detect or discriminate betweenthe presence of a number of different detectable substances due to thebroad spectrum emissions and emission tails of the labelling molecules.Another problem is that most dye molecules have a relatively narrowabsorption spectrum, thus requiring either multiple excitation beamsused either in tandem or sequentially for multiple wavelength probes, orelse a broad spectrum excitation source which is sequentially used withdifferent filters for sequential excitation of a series of probesrespectively excited at different wavelengths.

[0007] Another problem frequently encountered with existing dye moleculelabels is that of photostability. Available fluorescent moleculesbleach, or irreversibly cease to emit light, under repeated excitation(10⁴-10⁸) cycles of absorption/emission. These problems are oftensurmounted by minimizing the amount of time that the sample is exposedto light, and by removing oxygen and/or other radical species from thesample.

[0008] In addition, the probe tools used for the study of these systemsby electron microscopy techniques are completely different from theprobes used for study by fluorescence. Thus, it is not possible to labela material with a single type of probe for both electron microscopy andfor fluorescence.

[0009] It would, therefore, be desirable to provide a stable probematerial for biological applications having a wide absorption band andcapable of exhibiting either a detectable change in absorption or ofemitting radiation in a narrow wavelength band, without the presence ofthe large red emission tails characteristic of dye molecules (therebypermitting the simultaneous use of a number of such probe materials,each emitting light of a different narrow wavelength band) and/orcapable of scattering or diffracting radiation. It would also be equallydesirable to provide a single, stable probe material which can be usedto image the same sample by both light and electron microscopy.

SUMMARY OF THE INVENTION

[0010] The invention comprises a luminescent semiconductor nanocrystalcompound capable of linking to an affinity molecule to form an organoluminescent semiconductor nanocrystal probe capable of luminescenceand/or absorption and/or scattering or diffracting when excited by anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam, and capable of exhibiting a detectable change inabsorption and/or of emitting radiation in a narrow wavelength bandand/or scattering or diffracting when so excited. The luminescentsemiconductor nanocrystal compound preferably comprises: (1) asemiconductor nanocrystal capable of luminescence and/or absorptionand/or scattering or diffraction when excited by an electromagneticradiation source (of broad or narrow bandwidth) or a particle beam, andcapable of exhibiting a detectable change in absorption and/or ofemitting radiation in a narrow wavelength band and/or scattering ordiffracting when excited; and (2) a linking agent having a first portionlinked to the semiconductor nanocrystal, and a second portion capable oflinking to an affinity molecule.

[0011] The invention further comprises an organo luminescentsemiconductor nanocrystal probe formed by linking the above describedluminescent semiconductor nanocrystal compound to an affinity moleculecapable of bonding to a detectable substance in a material. As a resultthe organo luminescent semiconductor nanocrystal probe, in oneembodiment, is capable of absorbing or scattering or diffracting energyfrom either a particle beam or an electromagnetic radiation source (ofbroad or narrow bandwidth), and is capable of emitting electromagneticradiation in a narrow wavelength band when so excited; while in anotherembodiment the amount of energy so absorbed, or scattered, or diffractedfrom either a particle beam or an electromagnetic radiation source (ofbroad or narrow bandwidth), is detectable, i.e., the change inabsorption, scattering, or diffraction is detectable.

[0012] Therefore, treatment of a material with the organo luminescentsemiconductor nanocrystal probe, and subsequent exposure of this treatedmaterial to excitation energy (from either a particle beam or anelectromagnetic radiation source of broad or narrow bandwidth) todetermine the presence of the detectable substance within the material,will excite the semiconductor nanocrystals in the organo luminescentsemiconductor nanocrystal probe bonded to the detectable substance,resulting in the emission of electromagnetic radiation of a narrowwavelength band and/or a detectable change in the amount of energy beingabsorbed and/or scattered or diffracted, signifying the presence, in thematerial, of the detectable substance bonded to the organo luminescentsemiconductor nanocrystal probe.

[0013] The invention also comprises a process for making the luminescentsemiconductor nanocrystal compound and for making the organo luminescentsemiconductor nanocrystal probe comprising the luminescent semiconductornanocrystal compound linked to an affinity molecule capable of bondingto a detectable substance. The organo luminescent semiconductornanocrystal probe of the invention is stable with respect to repeatedexcitation by light, or exposure to oxygen or other radicals. Theinvention further comprises a process for treating a material, such as abiological material, to determine the presence of a detectable substancein the material which comprises contacting the material with the organoluminescent semiconductor nanocrystal probe, removing from the materialportions of the organo luminescent semiconductor nanocrystal probe notbonded to the detectable substance, and then exposing the material toactivation energy from either an electromagnetic radiation source (ofbroad or narrow bandwidth) or a particle beam. The presence of thedetectable substance in the material is then determined either bymeasuring the absorption of energy by the organo luminescentsemiconductor nanocrystal probe and/or detecting the emission ofradiation of a narrow wavelength band by the organo luminescentsemiconductor nanocrystal probe and/or detecting the scattering ordiffraction by the organo luminescent semiconductor nanocrystal probe,indicative (in either case) of the presence of the organo luminescentsemiconductor nanocrystal probe bonded to the detectable substance inthe material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a block diagram of the luminescent semiconductornanocrystal compound of the invention.

[0015]FIG. 2 is a block diagram of the organo luminescent semiconductornanocrystal probe of the invention.

[0016]FIG. 3 is a block diagram showing the affinity between adetectable substance and the organo luminescent semiconductornanocrystal probe of the invention.

[0017]FIG. 4 is a flow sheet illustrating the process of forming theorgano luminescent semiconductor nanocrystal probe of the invention.

[0018]FIG. 5 is a flow sheet illustrating a typical use of the organoluminescent semiconductor nanocrystal probe of the invention indetecting the presence of a detectable substance in a material such as abiological material.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention comprises a luminescent semiconductor nanocrystalcompound capable of linking to an organic molecule and capable ofexhibiting a detectable change in absorption and/or of emittingelectromagnetic radiation in a narrow wavelength band and/or scatteringor diffracting when excited by either an electromagnetic radiationsource (of broad or narrow bandwidth) or a particle beam. Theluminescent semiconductor nanocrystal compound, in turn, comprises: (1)semiconductor nanocrystals capable of exhibiting a detectable change inabsorption and/or of emitting electromagnetic radiation in a narrowwavelength band when excited by either an electromagnetic radiationsource (of broad or narrow bandwidth) or a particle beam; and (2) one ormore linking agents each having a first portion linked to thesemiconductor nanocrystal and a second portion capable of linking to anorganic affinity molecule.

[0020] The invention also comprises the above described luminescentsemiconductor nanocrystal compound linked to the organic affinitymolecule (through the linking agent) to form an organo luminescentsemiconductor nanocrystal probe capable of bonding to a detectablesubstance and capable of exhibiting a detectable change in absorptionand/or of emitting electromagnetic radiation in a narrow wavelength bandand/or scattering or diffracting when excited by either anelectromagnetic radiation source (of broad or narrow bandwidth) or aparticle beam. Treatment of a material (typically a biological material)with the organo luminescent semiconductor nanocrystal probe, andsubsequent exposure of this treated material to excitation energy, asdescribed above, to determine the presence of the detectable substancewithin the material, will excite the semiconductor nanocrystal in theorgano luminescent semiconductor nanocrystal probe bonded to thedetectable substance, causing the detectable change in absorption and/oremission of electromagnetic radiation of a narrow wavelength band and/orscattering or diffraction signifying (in either instance) the presencein the material, of the detectable substance bonded to the organoluminescent semiconductor nanocrystal probe.

[0021] The invention also comprises a process for making the luminescentsemiconductor nanocrystal compound, and a process for making the organoluminescent semiconductor nanocrystal probe comprising the luminescentsemiconductor nanocrystal compound linked to an affinity moleculecapable of bonding to a detectable substance.

[0022] The invention further comprises a process for treating amaterial, such as a biological material, to determine the presence of adetectable substance in the material which comprises: (1) contacting thematerial with the organo luminescent semiconductor nanocrystal probe,(2) removing from the material portions of the organo luminescentsemiconductor nanocrystal probe not bonded to the detectable substance,(3) exposing the material to energy (such as the above-describedelectromagnetic energy source or particle beam) capable of exciting thesemiconductor nanocrystal to cause a detectable change in absorptionand/or emission of electromagnetic radiation of a narrow wavelength bandand/or scattering or diffraction signifying (in either instance) thepresence of the organo luminescent semiconductor nanocrystal probebonded to the detectable substance in the material, and (4) detectingeither the change in absorbed energy or the electromagnetic radiationemitted or the scattering or diffraction by the semiconductornanocrystal in the organo luminescent semiconductor nanocrystal probe.

a. Definitions

[0023] By use of the terms “nanometer crystal” or “nanocrystal” hereinis meant an organic or inorganic single crystal particle having anaverage cross-section no larger than about 20 nanometers (nm) or 20×10⁻⁹meters (200 Angstroms), preferably no larger than about 10 nm (100Angstroms) and a minimum average cross-section of about 1 nm, althoughin some instances a smaller average cross-section nanocrystal, i.e.,down to about 0.5 nm (5 Angstroms), may be acceptable. Typically thenanocrystal will have an average cross-section ranging in size fromabout 1 nm (10 Angstroms) to about 10 nm (100 angstroms).

[0024] By use of the term “semiconductor nanocrystal” is meant ananometer crystal or nanocrystal of Group II-VI and Group III-Vsemiconductor compounds capable of emitting electromagnetic radiationupon excitation, although the use of Group IV semiconductors such asgermanium or silicon, or the use of organic semiconductors, may befeasible under certain conditions.

[0025] By use of the term “a narrow wavelength band”, with regard to theelectromagnetic radiation emission of the semiconductor nanocrystal, ismeant a wavelength band of emissions not exceeding about 40 nm, andpreferably not exceeding about 20 nm in width and symmetric about thecenter, in contrast to the emission bandwidth of about 100 nm for atypical dye molecule, with a red tail which may extend the band widthout as much as another 100 nm. It should be noted that the bandwidthsreferred to are determined from measurement of the width of theemissions at half peak height (FWHM), and are appropriate in the rangeof 200 nm to 2000 nm.

[0026] By use of the term “a broad absorption band”, with regard to theelectromagnetic radiation absorption of the semiconductor nanocrystal ismeant a continuously increasing absorption from the onset, which occursnear to, but at slightly higher energy than the “narrow wavelength band”of the emission. This is in contrast to the “narrow absorption band” ofdye molecules which occurs near the emission peak on the high energyside, but drops off rapidly away from that wavelength.

[0027] By use of the term “detectable substance” is meant an entity orgroup, the presence or absence of which in a material such as abiological material, is to be ascertained by use of theorgano-luminescent semiconductor nanocrystal probe of the invention.

[0028] By use of the term “affinity molecule” is meant the portion ofthe organo luminescent semiconductor nanocrystal probe of the inventionwhich will selectively bond to a detectable substance (if present) inthe material (e.g., biological material) being analyzed.

[0029] By use of the term “linking agent” is meant a substance capableof linking with a semiconductor nanocrystal and also capable of linkingto an affinity molecule.

[0030] The terms “link” and “linking” are meant to describe theadherence between the affinity molecule and the semiconductornanocrystals, either directly or through a moiety identified herein as alinking agent. The adherence may comprise any sort of bond, including,but not limited to, covalent, ionic, hydrogen bonding, Van der Waals'forces, or mechanical bonding, etc.

[0031] The terms “bond” and “bonding” are meant to describe theadherence between the affinity molecule and the detectable substance.The adherence may comprise any sort of bond, including, but not limitedto, covalent, ionic, or hydrogen bonding, Van der Waals' forces, ormechanical bonding, etc.

[0032] The term “luminescent semiconductor nanocrystal compound”, asused herein, is intended to define a semiconductor nanocrystal linked toone or more linking agents and capable of linking to an affinitymolecule, while the term “organo-luminescent semiconductor nanocrystalprobe” is intended to define a luminescent semiconductor nanocrystalcompound linked to an affinity molecule.

[0033] The term “glass” as used herein is intended to include one ormore oxides of silicon, boron, and/or phosphorus, or a mixture thereof,as well as the further optional inclusion of one or more metalsilicates, metal borates or metal phosphates therein.

b. The Semiconductor Nanocrystals

[0034] The semiconductor nanocrystals useful in the practice of theinvention include nanocrystals of Group II-VI semiconductors such asMgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS,ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe; and nanocrystals ofGroup III-V semiconductors such as GaAs, InGaAs, InP, and InAs. Asmentioned above, the use of Group IV semiconductors such as germanium orsilicon, or the use of organic semiconductors, may also be feasibleunder certain conditions.

[0035] Formation of nanometer crystals of Group III-V semiconductors isdescribed in copending and commonly assigned Alivisatos et al. Ser. No.08/235,265, filed Apr. 29, 1994 as an FWC application of Ser. No.07/796,246, filed Nov. 11, 1991; and Alivisatos et al. U.S. Pat. No.5,262,357, which also describes the formation of Group II-VIsemiconductor nanocrystals, and which is also assigned to the assigneeof this invention. Also described therein is the control of the size ofthe semiconductor nanocrystals during formation using crystal growthterminators. The teachings of Alivisatos et al. Ser. No. 08/235,265, andAlivisatos et al. U.S. Pat. No. 5,262,357 are each hereby specificallyincorporated by reference.

[0036] In a preferred embodiment, the nanocrystals are used in acore/shell configuration wherein a first semiconductor nanocrystal formsa core ranging in diameter, for example, from about 20 Å to about 100 Å,with a shell of another semiconductor nanocrystal material grown overthe core nanocrystal to a thickness of, for example, 1-10 monolayers inthickness. When, for example, a 1-10 monolayer thick shell of CdS isepitaxially grown over a core of CdSe, there is a dramatic increase inthe room temperature photoluminescence quantum yield. Formation of suchcore/shell nanocrystals is described more fully in a publication by oneof us with others entitled “Epitaxial Growth of Highly LuminescentCdSe/CdS Core/Shell Nanocrystals with Photostability and ElectronicAccessibility”, by Peng, Schlamp, Kadavanich, and Alivisatos, publishedin the Journal of the American Chemical Society, Volume 119, No. 30.1997, at pages 7019-7029, the subject matter of which is herebyspecifically incorporated herein by reference.

[0037] The semiconductor nanocrystals used in the invention will have acapability of emitting light within a narrow wavelength band of about 40nm or less, preferably about 20 nm or less, thus permitting thesimultaneous use of a plurality of differently colored organoluminescent semiconductor nanocrystal probes with differentsemiconductor nanocrystals without overlap (or with a small amount ofoverlap) in wavelengths of emitted light (unlike the use of dyemolecules with broad emission lines (e.g., ˜100 nm) and broad tails ofemission (e.g., another 100 nm) on the red side of the spectrum), thusallowing for the simultaneous detection of a plurality of detectablesubstances.

c. Affinity Molecule

[0038] The particular affinity molecule forming a part of theorgano-luminescent semiconductor nanocrystal probe of the invention willbe selected based on its affinity for the particular detectablesubstance whose presence or absence, for example, in a biologicalmaterial, is to be ascertained. Basically, the affinity molecule maycomprise any molecule capable of being linked to a luminescentsemiconductor nanocrystal compound which is also capable of specificrecognition of a particular detectable substance. In general, anyaffinity molecule useful in the prior art in combination with a dyemolecule to provide specific recognition of a detectable substance willfind utility in the formation of the organo-luminescent semiconductornanocrystal probes of the invention. Such affinity molecules include, byway of example only, such classes of substances as monoclonal andpolyclonal antibodies, nucleic acids (both monomeric and oligomeric),proteins, polysaccharides, and small molecules such as sugars, peptides,drugs, and ligands. Lists of such affinity molecules are available inthe published literature such as, by way of example, the “Handbook ofFluorescent Probes and Research Chemicals”, (sixth edition) by R. P.Haugland, available from Molecular Probes, Inc.

d. The Linking Agent

[0039] The organo-luminescent semiconductor nanocrystal probe of theinvention will usually find utility with respect to the detection of oneor more detectable substances in organic materials, and in particular tothe detection of one or more detectable substances in biologicalmaterials. This requires the presence, in the organo-luminescentsemiconductor nanocrystal probe, of an affinity molecule or moiety, asdescribed above, which will bond the organo-luminescent semiconductornanocrystal probe to the detectable substance in the organic/biologicalmaterial so that the presence of the detectable material may besubsequently ascertained. However, since the semiconductor nanocrystalsare inorganic, they may not bond directly to the organic affinitymolecule. In these case therefore, there must be some type of linkingagent present in the organo-luminescent semiconductor nanocrystal probewhich is capable of forming a link to the inorganic semiconductornanocrystal as well as to the organic affinity molecule in theorgano-luminescent semiconductor nanocrystal probe.

[0040] One form in which the semiconductor nanocrystal may be linked toan affinity molecule via a linking agent is by coating the semiconductornanocrystal with a thin layer of glass, such as silica (SiO_(x) wherex=1-2), using a linking agent such as a substituted silane, e.g.,3-mercaptopropyl-trimethoxy silane to link the nanocrystal to the glass.The glass-coated semiconductor nanocrystal may then be further treatedwith a linking agent, e.g., an amine such as3-aminopropyl-trimethoxysilane, which will function to link theglass-coated semiconductor nanocrystal to the affinity molecule. Thatis, the glass-coated semiconductor nanocrystal may then be linked to theaffinity molecule. It is within the contemplation of this invention thatthe original luminescent semiconductor nanocrystal compound may also bechemically modified after it has been made in order to link effectivelyto the affinity molecule. A variety of references summarize the standardclasses of chemistry which may be used to this end, in particular the“Handbook of Fluorescent Probes and Research Chemicals”, (6th edition)by R. P. Haugland, available from Molecular Probes, Inc., and the book“Bioconjugate Techniques”, by Greg Hermanson, available from AcademicPress, New York.

[0041] When the semiconductor nanocrystal is coated with a thin layer ofglass, the glass, by way of example, may comprise a silica glass(SiO_(x) where x=1-2), having a thickness ranging from about 0.5 nm toabout 10 nm, and preferably from about 0.5 nm to about 2 nm.

[0042] The semiconductor nanocrystal is coated with the coating of thinglass, such as silica, by first coating the nanocrystals with asurfactant such as tris-octyl-phosphine oxide, and then dissolving thesurfactant-coated nanocrystals in a basic methanol solution of a linkingagent, such as 3-mercaptopropyl-tri-methoxy silane, followed by partialhydrolysis which is followed by addition of a glass-affinity moleculelinking agent such as amino-propyl trimethoxysilane which will link tothe glass and serve to form a link with the affinity molecule.

[0043] When the linking agent does not involve the use of a glasscoating on the semiconductor nanocrystal, it may comprise a number ofdifferent materials, depending upon the particular affinity molecule,which, in turn, depends upon the type of detectable material beinganalyzed for. It should also be noted that while an individual linkingagent may be used to link to an individual semiconductor nanocrystal, itis also within the contemplation of the invention that more than onelinking agent may bond to the same semiconductor nanocrystal and viceversa.

[0044] A few examples of the types of linking agents which may be usedto link to both the semiconductor nanocrystal (or to a glass coating onthe nanocrystal) and to the organic affinity molecule in the probe areillustrated in the table below, it being understood that this is notintended to be an exhaustive list: Linking Agent Structure Name

N-(3-aminopropyl)3-mercapto-benzamide

3-aminopropyl-trimethoxysilane

3-mercaptopropyl-trimethoxysilane

3-maleimidopropyl-trimethoxysilane

3-hydrazidopropyl-trimethoxysilane

[0045] It should be further noted that a plurality of polymerizablelinking agents may be used together to form an encapsulating net orlinkage around an individual nanocrystal (or group of nanocrystals).This is of particular interest where the particular linking agent isincapable of forming a strong bond with the nanocrystal. Examples oflinking agents capable of bonding together in such a manner to surroundthe nanocrystal with a network of linking agents include, but are notlimited to: diacetylenes, acrylates, acrylamides, vinyl, styryl, and theaforementioned silicon oxide, boron oxide, phosphorus oxide, silicates,borates and phosphates.

e. The Excitation of the Probe and Detection of Emission/Absorption

[0046] As previously mentioned, the organo luminescent semiconductornanocrystal probe of the invention is capable of being excited over abroad bandwidth, yet exhibits emission in a narrow wavelength band, incontrast to the dye molecules used in the prior art. Thuselectromagnetic radiation of wavelength ranging from x-ray toultraviolet to visible to infrared waves may be used to excite theluminescent semiconductor nanocrystals in the probe. In addition, theluminescent semiconductor nanocrystals are capable of excitation frombombardment with a particle beam such as an electron beam (e-beam).Furthermore, because of the broad bandwidth at which the luminescentsemiconductor nanocrystals are excitable, one may use a commonexcitation source for the simultaneous excitation of several probes,i.e., several probes which give off radiation at different frequencies,thus permitting simultaneous excitation and detection of the presence ofseveral probes indicating, for example, the presence of severaldetectable substances in the material being examined.

[0047] Thus, for example, a laser radiation source of a given frequency,e.g., blue light, may be used to excite a first organo luminescentsemiconductor nanocrystal probe capable of emitting radiation of asecond frequency, e.g., red light, indicating the presence, in thematerial being illuminated, of a first detectable substance to which theparticular red light-emitting organo luminescent semiconductornanocrystal probe has bonded. At the same time, the same blue lightlaser source may also be exciting a second organo luminescentsemiconductor nanocrystal probe (in the same material) capable ofemitting radiation of a third frequency, e.g., green light, indicatingthe presence, in the material being illuminated, of a second detectablesubstance to which the particular green light-emitting organoluminescent semiconductor nanocrystal probe has bonded. Thus, unlike theprior art, multiple excitation sources need not be used (because of thebroad bandwidth in which the organo luminescent semiconductornanocrystal probe of the invention is capable of being excited), and thenarrow band of emission of the specific semiconductor nanocrystals ineach probe makes possible the elimination of sequencing and/or elaboratefiltering to detect the emitted radiation.

[0048] With respect to the absorption of energy by the probe of theinvention, when the excitation source is an electron beam, or an X-raysource, the presence of the organo luminescent semiconductor nanocrystalprobe bonded to the detectable substance of interest in the materialbeing analyzed can be ascertained using a commercially available energyabsorption or scattering or diffraction detection system wherein changesin absorption or scattering cross section or in diffraction of thematerial being analyzed can be detected, signifying the presence of theprobe in the material, which, in turn, indicates the presence of thedetectable substance to which the probe is bonded in the material beinganalyzed. In addition, it may be possible to use electron or X-raysources to detect the presence of the organo luminescent semiconductornanocrystal probe bonded to the detectable substance by using aconventional detection system for the emission of visible light toobserve the visible emission in the narrow wavelength of emission of theprobe.

[0049] The following examples will serve to further illustrate theformation of the organo luminescent semiconductor nanocrystal probes ofthe invention, as well as their use in detecting the presence of adetectable substance in a material such as a biological material.

EXAMPLE 1

[0050] To illustrate the formation of the luminescent semiconductornanocrystal compound (comprising the semiconductor nanocrystals linkedto a linking agent) 20 ml. of a 5 mM solution of (4-mercapto)benzoicacid was prepared with a pH of 10 using (CH₃)₄NOH.5H₂O. 20 mg oftris-octylphosphine oxide coated CdSe/CdS core/shell nanocrystals wereadded to the solution and stirred until completely dissolved. Theresultant nanocrystal/linking agent solution was heated for 5 hours at50-60° C. and then concentrated to a few ml by evaporation. Then anequal volume of acetone was added and the nanocrystals precipitate outof solution homogeneously. The precipitate was then washed with acetone,dried, and then can be stored.

[0051] The luminescent semiconductor nanocrystal compound prepared abovecan be linked with an appropriate affinity molecule to form the organoluminescent semiconductor nanocrystal probe of the invention to treat abiological material to determine the presence or absence of a detectablesubstance. That is, the luminescent semiconductor nanocrystal compoundprepared above can be linked, for example, with avidin or streptavidin(as the affinity molecule) to form an organo luminescent semiconductornanocrystal probe to treat a biological material to ascertain thepresence of biotin; or the luminescent semiconductor nanocrystalcompound prepared above can be linked with anti-digoxiginen to form anorgano luminescent semiconductor nanocrystal probe to treat a biologicalmaterial to ascertain the presence of digoxiginen.

EXAMPLE 2

[0052] To illustrate the formation of luminescent semiconductornanocrystal compound (comprising glass-coated semiconductor nanocrystalslinked to a linking agent), 50 μl of 3-mercaptopropyl-trimethoxy silanewas added to 40 ml of an anhydrous solution of 25 vol. %dimethylsulfoxide in methanol, and the pH was adjusted to 10-11 using(CH₃)₄NOH.5H₂O. 10 mg of tris-octylphosphine oxide coated CdSe/CdScore-shell particles, prepared by the technique described in theaforementioned Peng, Schlamp, Kadavanich, and Alivisatos article, werethen dissolved in this solution, and stirred for several hours. Thesolution was diluted with 40 ml of methanol adjusted to a pH of 10 with(CH₃)₄NOH.5H₂O, and heated for 1 hour at 69° C. The solution was stirredfor an hour, and 40 ml of a 90 vol. % methanol/9.89 vol. % H₂O/0.1 vol.% trimethoxysilylpropyl urea/0.01 vol. % aminopropyl-trimethoxy silanesolution which had been stirring for at least an hour, was added, andstirred for 2 hours. Subsequently the reaction was heated to 69° C. for15 minutes, and then cooled. 10 ml of a 10 vol. % chlorotrimethyl silanesolution in methanol which had been adjusted to a pH of 10 using(CH₃)₄NOH.5H₂O was added, stirred for 2 hours, then heated to 60° C. andthen partially concentrated under vacuum. Once the methanol had allevaporated, the solution was precipitated with acetone as an oil productcomprising the luminescent semiconductor nanocrystal compound. Theluminescent semiconductor nanocrystal compound may then be redissolvedin water, and in a variety of buffer solutions to prepare it for linkingit to an affinity molecule to form the organo luminescent semiconductornanocrystal probe of the invention to treat a biological material todetermine the presence or absence of a detectable substance.

[0053] Thus, the invention provides an organo luminescent semiconductornanocrystal probe containing a semiconductor nanocrystal capable, uponexcitation by either electromagnetic radiation (of either narrow orbroad bandwidth) or particle beam, of emitting electromagnetic radiationin a narrow wavelength band and/or absorbing energy and/or scattering ordiffracting said excitation, thus permitting the simultaneous usage of anumber of such probes emitting different wavelengths of electromagneticradiation to thereby permit simultaneous detection of the presence of anumber of detectable substances in a given material. The probe materialis stable in the presence of light or oxygen, capable of being excitedby energy over a wide spectrum, and has a narrow band of emission,resulting in an improved material and process for the simultaneousand/or sequential detection of a number of detectable substances in amaterial such as a biological material.

Having thus described the invention what is claimed is:
 1. A luminescentsemiconductor nanocrystal compound capable of linking to an affinitymolecule and capable of emitting electromagnetic radiation in a narrowwavelength band when excited comprising: a) a semiconductor nanocrystalcapable of emitting light in a narrow wavelength band when excited; andb) at least one linking agent linked to said semiconductor nanocrystaland capable of linking to said affinity molecule.
 2. The luminescentsemiconductor nanocrystal compound of claim 1 wherein said semiconductornanocrystal is capable of absorbing energy over a wide bandwidth.
 3. Theluminescent semiconductor nanocrystal compound of claim 1 wherein saidlinking agent includes a glass coating on said semiconductor nanocrystalcapable of being linked to said affinity molecule through a furtherlinking agent capable of linking to both said glass coating and saidaffinity molecule.
 4. The luminescent semiconductor nanocrystal compoundof claim 1 wherein said glass coating on said semiconductor nanocrystalcomprises a coating of silica glass.
 5. The luminescent semiconductornanocrystal compound of claim 1 wherein said linking agent comprise afirst portion linked to said semiconductor nanocrystal and a secondportion capable of linking to said affinity molecule.
 6. The luminescentsemiconductor nanocrystal compound of claim 1 wherein said one or morelinking agents comprises a glass coating on said semiconductornanocrystal and a linking material having a first portion linked to saidglass coating on said semiconductor nanocrystal and a second portioncapable of linking to said affinity molecule.
 7. An organo luminescentsemiconductor nanocrystal probe capable of bonding with a detectablesubstance and capable of emitting electromagnetic radiation in a narrowwavelength band when excited, comprising a luminescent semiconductornanocrystal compound linked to an affinity molecule capable of bondingto said detectable substance.
 8. An organo luminescent semiconductornanocrystal probe capable of bonding with a detectable substance andcapable of emitting electromagnetic radiation in a narrow wavelengthband when excited comprising: a) a semiconductor nanocrystal capable ofemitting electromagnetic radiation in a narrow wavelength band whenexcited; b) at least one linking agent linked to said semiconductornanocrystal and having a second portion capable of linking to anaffinity molecule; and c) an affinity molecule linked to said secondportion of said linking agent, and capable of selectively bonding tosaid detectable substance; whereby treatment of a material with saidorgano luminescent semiconductor nanocrystal probe, and subsequentexposure of said treated material to excitation energy to determine thepresence of said detectable substance within said material will excitesaid semiconductor nanocrystal in said organo luminescent semiconductornanocrystal probe bonded to said detectable substance causing theemission of electromagnetic radiation of a narrow wavelength bandsignifying the presence, in said material, of said detectable substancebonded to said organo luminescent semiconductor nanocrystal probe. 9.The organo luminescent semiconductor nanocrystal probe of claim 8wherein said linking agent comprises a glass coating on saidsemiconductor nanocrystal.
 10. The organo luminescent semiconductornanocrystal probe of claim 8 wherein said material treated with saidorgano luminescent semiconductor nanocrystal probe to determine thepresence of said detectable substance comprises a biological material.11. The organo luminescent semiconductor nanocrystal probe of claim 8wherein said material treated with said organo luminescent semiconductornanocrystal probe to determine the presence of said detectable substancecomprises an organic material.
 12. The organo luminescent semiconductornanocrystal probe of claim 8 wherein said material treated with saidorgano luminescent semiconductor nanocrystal probe to determine thepresence of said detectable substance comprises an inorganic material.13. A process for forming a luminescent semiconductor nanocrystalcompound capable of linking to an affinity molecule and capable ofemitting electromagnetic radiation electromagnetic radiation in a narrowwavelength band when excited which comprises: linking together asemiconductor nanocrystal capable of emitting electromagnetic radiationin a narrow wavelength band when excited and a linking agent having afirst portion linked to said semiconductor nanocrystal and a secondportion capable of linking to an affinity molecule.
 14. The process ofclaim 13 which further comprises forming a glass coating on saidsemiconductor nanocrystals and then treating said glass with a linkingagent capable of linking with an affinity molecule
 15. A process forforming an organo luminescent semiconductor nanocrystal probe capable ofbonding with a detectable substance and capable of emittingelectromagnetic radiation in a narrow wavelength band when excited whichcomprises linking a luminescent semiconductor nanocrystal compound withan affinity molecule capable of bonding with a detectable substance. 16.A process for forming an organo luminescent semiconductor nanocrystalprobe capable of bonding with a detectable substance and capable ofemitting electromagnetic radiation in a narrow wavelength band whenexcited which comprises the steps of: a) linking a semiconductornanocrystal capable of emitting electromagnetic radiation in a narrowwavelength band when excited with a linking agent having a first portionlinked to said semiconductor nanocrystal and a second portion capable oflinking to an affinity molecule; and b) linking said linking agent andan affinity molecule capable of bonding with said detectable substance.17. The process of claim 16 wherein said step of linking together saidsemiconductor nanocrystal and said linking agent is carried out prior tosaid step of linking together said linking agent and said affinitymolecule.
 18. The process of claim 16 wherein said step of linkingtogether said linking agent and said more affinity molecule is carriedout prior to said step of linking together said semiconductornanocrystal and said linking agent.
 19. The process of claim 16 whereinsaid step of linking together said semiconductor nanocrystal and saidlinking agent further comprises coating said semiconductor nanocrystalwith a glass and then treating said glass-coated semiconductornanocrystal with a linking agent capable of linking to said affinitymolecule.
 20. A process for treating a material to determine thepresence of one or more detectable substances in said material whichcomprises: a) contacting said material with a first organo luminescentsemiconductor nanocrystal probe capable of bonding with a firstdetectable substance, if present, in said material, and capable ofemitting electromagnetic radiation in a first narrow wavelength bandwhen excited, said first organo luminescent semiconductor nanocrystalprobe comprising: i) a first semiconductor nanocrystal capable of beingexcited over a broad bandwidth and capable of emitting electromagneticradiation in said first narrow wavelength band when excited; ii) anaffinity molecule capable of selectively bonding to said detectablesubstance; and iii) a linking agent linked to said first semiconductornanocrystal and also linked to said affinity molecule; b) removing, fromsaid material, portions of said first organo luminescent semiconductornanocrystal probe not bonded to said first detectable substance; and c)exposing said material to energy capable of exciting said firstsemiconductor nanocrystal to emit electromagnetic radiation in saidfirst narrow wavelength band, indicative of the presence of said firstdetectable substance in said material; and d) detecting saidelectromagnetic radiation in said first narrow wavelength band emittedby said first semiconductor nanocrystal in said first organo luminescentsemiconductor nanocrystal probe.
 21. The process of claim 20 whichincludes the further step of treating said material with at least asecond organo luminescent semiconductor nanocrystal probe capable ofbonding to an additional detectable substance in said material, andcontaining a second semiconductor nanocrystal capable of being excitedover a broad bandwidth and capable of emitting electromagnetic radiationin a second narrow wavelength band different from said first narrowwavelength band, whereby the exposure of said material to energy capableof exciting both said first and second nanocrystals will cause any ofsaid first or second semiconductor nanocrystals present in said materialto emit electromagnetic radiation of differing narrow wavelength bands,whereby the presence or absence of more than one detectable substance ina material may be simultaneously detected using a single excitationenergy source.
 22. The process of claim 21 wherein at least one furtherorgano luminescent semiconductor nanocrystal probe is used to treat saidmaterial, with each of said organo luminescent semiconductor nanocrystalprobes selectively bondable to a different detectable substance and eachof said organo luminescent semiconductor nanocrystal probes capable ofbeing excited over a broad bandwidth and capable of emittingelectromagnetic radiation of a different narrow wavelength band, wherebya plurality of detectable substances may be simultaneously analyzed forin a material using a single excitation source.
 23. The process of claim21 wherein said material is treated with all of said organo luminescentsemiconductor nanocrystal probes prior to said step of removing, fromsaid material, portions of said first organo luminescent semiconductornanocrystal probe not bonded to said first detectable substance, andsaid step of removing further comprises removing portions of all of saidorgano luminescent semiconductor nanocrystal probes not bonded to adetectable substance in said material.
 24. The process of claims 21, 22,or 23, whereby the exposure of the material to light of a selectedwavelength is used to excite selectively one or more, but not all, ofsaid organo luminescent semiconductor nanocrystal probes, thus allowingidentification of the presence of specific labelled detectablesubstances, or subsets of different labelled detectable substances insaid material.
 25. The process for treating a material of claim 20wherein said material comprises a biological material.
 26. The processfor treating a material of claim 20 wherein said step of exposing saidmaterial to energy capable of exciting said first semiconductornanocrystal to emit electromagnetic radiation further comprises exposingsaid material to a source of electromagnetic radiation capable ofemitting photons of a broad or narrow spectrum.
 27. The process fortreating a material of claim 20 wherein said step of exposing saidmaterial to energy capable of exciting said first semiconductornanocrystal to emit electromagnetic radiation further comprises exposingsaid material to an electron beam.
 28. A process for treating a materialto determine the presence of a detectable substance in said materialwhich comprises: a) contacting said material with an organo luminescentsemiconductor nanocrystal probe capable of bonding with a firstdetectable substance, if present, in said material, and capable ofabsorbing energy when excited, said organo luminescent semiconductornanocrystal probe comprising: i) a semiconductor nanocrystal capable ofbeing excited over a broad bandwidth and capable of absorbing energywhen excited; ii) an affinity molecule capable of selectively bonding tosaid detectable substance; and iii) a linking agent linked to said firstsemiconductor nanocrystal and also linked to said affinity molecule; b)removing, from said material, portions of said organo luminescentsemiconductor nanocrystal probe not bonded to said first detectablesubstance; and c) exposing said material to energy capable of excitingsaid first semiconductor nanocrystal to absorb energy, indicative of thepresence of said first detectable substance in said material; and d)detecting the change in absorbed energy, indicative of the presence ofsaid organo luminescent semiconductor nanocrystal probe in said materialbonded to said detectable substance.
 29. The process of claim 28 whichincludes the further step of treating said material with at least asecond organo luminescent semiconductor nanocrystal probe capable ofbonding to an additional detectable substance in said material, andcontaining a second semiconductor nanocrystal capable of being excitedover a broad bandwidth resulting in a detectable change in absorbance,and whereby the exposure of said material to energy capable of excitingboth said first and second nanocrystals will cause any of said first orsecond semiconductor nanocrystals present in said material to absorbelectromagnetic radiation of differing wavelength bands, whereby thepresence or absence of more than one detectable substance in a materialmay be simultaneously detected using a single excitation energy source.30. The process of claim 29 wherein at least one further organoluminescent semiconductor nanocrystal probe is used to treat saidmaterial, with each of said organo luminescent semiconductor nanocrystalprobes selectively bondable to a different detectable substance and eachof said organo luminescent semiconductor nanocrystal probes capable ofbeing excited over a broad bandwidth and capable of absorbingelectromagnetic radiation, whereby a plurality of detectable substancesmay be simultaneously analyzed for in a material using a singleexcitation source.
 31. The process for treating a material of claim 28wherein said step of exposing said material to energy capable ofexciting said first semiconductor nanocrystal to emit electromagneticradiation further comprises exposing said material to a source ofelectromagnetic radiation capable of emitting photons of a broad ornarrow spectrum.
 32. The process for treating a material of claim 28wherein said step of exposing said material to energy capable ofexciting said first semiconductor nanocrystal to emit electromagneticradiation further comprises exposing said material to an X-ray source.33. A process for treating a material to determine the presence of adetectable substance in said material which comprises: a) contactingsaid material with an organo luminescent semiconductor nanocrystal probecapable of bonding with a first detectable substance, if present, insaid material, and capable of scattering or diffracting energy whenexcited, said organo luminescent semiconductor nanocrystal probecomprising: i) a semiconductor nanocrystal capable of scattering ordiffracting energy over a broad bandwidth with a characteristiccross-section; ii) an affinity molecule capable of selectively bondingto said detectable substance; and iii) a linking agent linked to saidfirst semiconductor nanocrystal and also linked to said affinitymolecule; b) removing, from said material, portions of said organoluminescent semiconductor nanocrystal probe not bonded to said firstdetectable substance; and c) exposing said material to energy capable ofexciting said first semiconductor nanocrystal to scatter or diffractenergy, indicative of the presence of said first detectable substance insaid material; and d) detecting the change in scattered or diffractedenergy, indicative of the presence of said organo luminescentsemiconductor nanocrystal probe in said material bonded to saiddetectable substance.
 34. The process of claim 33 which includes thefurther step of treating said material with at least a second organoluminescent semiconductor nanocrystal probe capable of bonding to asecond detectable substance in said material, and containing a secondsemiconductor nanocrystal also capable of scattering or diffractingenergy, resulting in a detectable change in scattering cross-section,and whereby the exposure of said material to energy capable ofscattering or diffracting from both said first and second nanocrystalswill cause any of said first or second semiconductor nanocrystalspresent in said material to scatter or diffract energy with scatteringcross sections characteristic of the particular organo luminescentsemiconductor nanocrystal probe, whereby the presence or absence of morethan one detectable substance in a material may be simultaneouslydetected using a single excitation energy source.
 35. The process ofclaim 34 wherein at least one further organo luminescent semiconductornanocrystal probe is used to treat said material, with each of saidorgano luminescent semiconductor nanocrystal probes selectively bondableto a different detectable substance and each of said organo luminescentsemiconductor nanocrystal probes exhibiting a different scattering crosssection and capable of scattering or diffracting energy, whereby aplurality of detectable substances may be simultaneously analyzed for ina material using a single excitation source.
 36. The process fortreating a material of claim 33 wherein said step of exposing saidmaterial to energy capable of exciting said first semiconductornanocrystal to scatter or diffract energy further comprises exposingsaid material to an electron beam or other particle beam.
 37. Theprocess for treating a material of claim 33 wherein said step ofexposing said material to energy capable of exciting said firstsemiconductor nanocrystal to scatter or diffract energy furthercomprises exposing said material to an X-ray source.
 38. The process fortreating a material of claim 33 wherein said step of exposing saidmaterials to energy capable of causing said first semiconductornanocrystal to scatter or diffract energy, and said step of detectingsaid scattering or diffraction of energy, are both carried out using atransmission electron microscope.
 39. The process for treating amaterial of claim 33 wherein said step of exposing said materials toenergy capable of causing said first semiconductor nanocrystal toscatter or diffract energy, and said step of detecting said scatteringor diffraction of energy, are both carried out using a scanning electronmicroscope.
 40. A luminescent semiconductor nanocrystal compound capableof linking to an affinity molecule and capable of absorbing energy in anarrow wavelength band when excited comprising: a) a semiconductornanocrystal capable of absorbing energy in a narrow wavelength band whenexcited; and b) at least one linking agent linked to said semiconductornanocrystal and capable of linking to said affinity molecule.
 41. Aluminescent semiconductor nanocrystal compound capable of linking to anaffinity molecule and capable of scattering or diffracting energy in anarrow wavelength band when excited comprising: a) a semiconductornanocrystal capable of scattering or diffracting energy in a narrowwavelength band when excited; and b) at least one linking agent linkedto said semiconductor nanocrystal and capable of linking to saidaffinity molecule.
 42. An organo luminescent semiconductor nanocrystalprobe capable of bonding with a detectable substance and capable ofabsorbing energy in a narrow wavelength band when excited, comprising aluminescent semiconductor nanocrystal compound linked to an affinitymolecule capable of bonding to said detectable substance.
 43. An organoluminescent semiconductor nanocrystal probe capable of bonding with adetectable substance and capable of absorbing energy in a narrowwavelength band when excited comprising: a) a semiconductor nanocrystalcapable of absorbing energy in a narrow wavelength band when excited; b)at least one linking agent linked to said semiconductor nanocrystal andhaving a second portion capable of linking to an affinity molecule; andc) an affinity molecule linked to said second portion of said linkingagent, and capable of selectively bonding to said detectable substance;whereby treatment of a material with said organo luminescentsemiconductor nanocrystal probe, and subsequent exposure of said treatedmaterial to excitation energy to determine the presence of saiddetectable substance within said material will excite said semiconductornanocrystal in said organo luminescent semiconductor nanocrystal probebonded to said detectable substance causing the absorption of energy ofa narrow wavelength band signifying the presence, in said material, ofsaid detectable substance bonded to said organo luminescentsemiconductor nanocrystal probe.
 44. An organo luminescent semiconductornanocrystal probe capable of bonding with a detectable substance andcapable of scattering or diffracting energy in a narrow wavelength bandwhen excited, comprising a luminescent semiconductor nanocrystalcompound linked to an affinity molecule capable of bonding to saiddetectable substance.
 45. An organo luminescent semiconductornanocrystal probe capable of bonding with a detectable substance andcapable of scattering or diffracting energy in a narrow wavelength bandwhen excited comprising: a) a semiconductor nanocrystal capable ofscattering or diffracting energy in a narrow wavelength band whenexcited; b) at least one linking agent linked to said semiconductornanocrystal and having a second portion capable of linking to anaffinity molecule; and c) an affinity molecule linked to said secondportion of said linking agent, and capable of selectively bonding tosaid detectable substance; whereby treatment of a material with saidorgano luminescent semiconductor nanocrystal probe, and subsequentexposure of said treated material to excitation energy to determine thepresence of said detectable substance within said material will excitesaid semiconductor nanocrystal in said organo luminescent semiconductornanocrystal probe bonded to said detectable substance causing thescattering or diffracting of energy of a narrow wavelength bandsignifying the presence, in said material, of said detectable substancebonded to said organo luminescent semiconductor nanocrystal probe.
 46. Aprocess for forming a luminescent semiconductor nanocrystal compoundcapable of linking to an affinity molecule and capable of absorbingenergy in a narrow wavelength band when excited which comprises: linkingtogether a semiconductor nanocrystal capable of absorbing energy in anarrow wavelength band when excited and a linking agent having a firstportion linked to said semiconductor nanocrystal and a second portioncapable of linking to an affinity molecule.
 47. A process for forming aluminescent semiconductor nanocrystal compound capable of linking to anaffinity molecule and capable of scattering or diffracting energy in anarrow wavelength band when excited which comprises: linking together asemiconductor nanocrystal capable of scattering or diffracting energy ina narrow wavelength band when excited and a linking agent having a firstportion linked to said semiconductor nanocrystal and a second portioncapable of linking to an affinity molecule.
 48. A process for forming anorgano luminescent semiconductor nanocrystal probe capable of bondingwith a detectable substance and capable of absorbing energy in a narrowwavelength band when excited which comprises linking a luminescentsemiconductor nanocrystal compound with an affinity molecule capable ofbonding with a detectable substance.
 49. A process for forming an organoluminescent semiconductor nanocrystal probe capable of bonding with adetectable substance and capable of absorbing energy in a narrowwavelength band when excited which comprises the steps of: a) linking asemiconductor nanocrystal capable of absorbing energy in a narrowwavelength band when excited with a linking agent having a first portionlinked to said semiconductor nanocrystal and a second portion capable oflinking to an affinity molecule; and b) linking said linking agent andan affinity molecule capable of bonding with said detectable substance.50. A process for forming an organo luminescent semiconductornanocrystal probe capable of bonding with a detectable substance andcapable of scatterin or diffracting energy in a narrow wavelength bandwhen excited which comprises linking a luminescent semiconductornanocrystal compound with an affinity molecule capable of bonding with adetectable substance.
 51. A process for forming an organo luminescentsemiconductor nanocrystal probe capable of bonding with a detectablesubstance and capable of scattering or diffracting energy in a narrowwavelength band when excited which comprises the steps of: a) linking asemiconductor nanocrystal capable of scattering or diffracting energy ina narrow wavelength band when excited with a linking agent having afirst portion linked to said semiconductor nanocrystal and a secondportion capable of linking to an affinity molecule; and b) linking saidlinking agent and an affinity molecule capable of bonding with saiddetectable substance.